Seed Preparation & Addition

Source

• Domestic WW supernatant; settled at 20° C > 1 hr but < 36 hr.
• Commercial seed (e.g., BOD seed, Polyseed)
• …may need to mix longer or differently than manufacturer recommends
• DO NOT mix seed in distilled or deionized water!

Adding a seed capsule to deionized water causes an osmotic imbalance that will kill seed organisms. The purified water--containing no salts-- rushes across the bacterial cell walls. The bacteria subsequently swell up and burst (or lyse) which, if it does not kill them, will severly impair their ability to utilize oxygen.

• Effluent from a biological treatment system is NOT recommended, as it creates the potential for nitrification to occur.

If you do use effluent from a biological treatment system (or recycle effluent) as your seed source, use of a nitrification inhibition is recommended.

Preparing seed

We recommend decanting and stirring vs. drawing individual aliquots off top. When you draw individual aliquots using a pipet, the vacuum action actually draws up some of the settled organisms, resulting in much more variable seed additions. By letting the seed settle and then carefully pouring off--or decanting-- the supernatant, you ensure a much more consistent (between additions) seed.

Back to Top

Polyseed™ hydration process

photos: George Bowman, Wisconsin State Laboratory of Hygiene

Back to Top

BOD Seed™ hydration process

photos: George Bowman, Wisconsin State Laboratory of Hygiene

Seed Addition

• You can either:
  • Seed: dilution water
  • samples directly?
• Either practice is acceptable
• Seeding dilution water ensures all samples seeded
• Seeding individual samples makes calculating seed correction a much easier task

Back to Top

Adding Seed Directly to Bottles

• Adjust amount of seed to BOD bottle to obtain GGA results in the 198 ± 30.5 range;
• Never pipet seed material into a dry BOD bottle.
• Always have some dilution water in first.
• Adding seed to DI water can rupture (lyse) cells!!!

Back to Top

2 mLs seed doesn’t always work!

Many labs use a standard 2 mLs of seed to seed samples or GGA. This may be becuase "it's what we've always done" or because that's what the manufacturer recommends. The most important thing to rememeber when deciding how much seed to add is to consider GGA results. The proper amount of seed to add is that volume of seed that yields acceptable GGA performance. The graph below clearly shows that seed strength varies with seed source, and 2 mLs of seed may not work for all seed sources… and may evenb change between lots obtained from a single vendor.

Data: Wisconsin State Laboratory of Hygiene

Back to Top

Seed Correction

Adding Seed Directly to Bottles

Bottle A is not used due to the insufficient final DO

Average the seed controls that meet depletion criteria
= (0.34 + 0.41) ÷ 2
= 0.375 mg/L DO depletion per mL seed

Thus, if 2 ml undiluted seed is added to each sample bottle,
seed correction = 0.375 mg/L DO X 2 ml seed = 0.75 mg/L

Therefore, 0.75 mg/L is subtracted from the depletion of each sample BOD depletion before calculating final BOD result.

Back to Top

Adding Seed to Dilution Water

When seeding dilution water, there is no need for seed controls per se…a 300 mL volume of dilution water is analyzed and THAT serves as the seed control.

In the following example, the dilution water "seed control", labeled "Dilution Water Blank" here, shows an oxygen depletion of 2.4 mg/L after 5 days.

For the sample, in dilution "1", we need to correct for the depletion due to dilution water itself. Since 300 mLs depleted 2.4 mg/L, then 150 mLs would be expected to deplete 150/300 or 0.5 x 2.4 mg/L, or 1.2 mg/L.

Depletion due to seed:
Sample Dilution 1 = 2.4 x (150/300) = 2.4 x 0.5 = 1.2
Sample Dilution 2 = 2.4 x (200/300) = 2.4 x 0.6667 = 1.6
Sample Dilution 3 = 2.4 x (250/300) = 2.4 x 0.8333 = 2.0

Average BOD of three dilutions =
= 12.4 + 12.6 + 13.8 ÷ 3
= 38.8 ÷ 3
= 12.9

The hardest part of choosing to seed dilution water is choosing how much seed to add to dilution water to allow enough depletion in diluted samples. For example…if 300 mLs of dilution water only depleted 1.0 mg/L, that would not be sufficient….the same rules as for samples apply. You need to add enough seed organisms to the dilution water such that 300 mLs produces a drop of at least 2.0 mg/L

The other thing to remember is that if dilution water is seeded, ALL dilutions must be individually adjusted for seed correction.

Back to Top

Seed Controls

The most critical thing to remember with seed controls is that the purpose of analyzing seed controls is to determine the BOD of your seed material. Therefore, seed controls MUST be subject to the same criteria as samples, including number of dilutions, and depletion criteria.

• Need at least 2 dilutions
• Best to do 3 dilutions
• Calculate seed correction factor
• Should deplete between 0.6 to 1.0 mg/L
• Standard Methods has changed its position on this
• Less emphasis on Seed Control; More on GGA

Back to Top

Seeding Summary

• It has been reported that commercial seeds yield low GGA results. Good GGA results were achieved when larger volumes of the commercial seed was used.
• The (Standard Methods) recommended seed control depletion criteria of 0.6 to 1.0 mg/l were exceeded in order to achieve better results for the GGA.
• It's GUIDANCE!
  

  Many states and auditors continue to view the seed depletion criteria of 0.6 to 1.0 mg/L as a requirement. This is simply not the case. Standard Methods always used the word "should" and furthermore has since upgraded the language to clarify this issue. The intent here is to adjust seed volume to the point at which GGA acceptance criteria are met. If GGA results are low, you should add more seed. If GGA results are too high, reduce the seed volume and/or evaluate potential sources of contamination.

• Domestic primary wastewater seed seemed to provide the most consistent GGA results.
• Note however that ANY wastewater seed strength can change with conditions (e.g.; rainfall, I&I).

Back to Top